Bis-enoxacin blocks rat alveolar bone resorption from experimental periodontitis

Abstract

Periodontal diseases are multifactorial, caused by polymicrobial subgingival pathogens, including Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia. Chronic periodontal infection results in inflammation, destruction of connective tissues, periodontal ligament, and alveolar bone resorption, and ultimately tooth loss. Enoxacin and a bisphosphonate derivative of enoxacin (bis-enoxacin) inhibit osteoclast formation and bone resorption and also contain antibiotic properties. Our study proposes that enoxacin and/or bis-enoxacin may be useful in reducing alveolar bone resorption and possibly bacterial colonization. Rats were infected with 10(9) cells of polymicrobial inoculum consisting of P. gingivalis, T. denticola, and T. forsythia, as an oral lavage every other week for twelve weeks. Daily subcutaneous injections of enoxacin (5 mg/kg/day), bis-enoxacin (5, 25 mg/kg/day), alendronate (1, 10 mg/kg/day), or doxycycline (5 mg/day) were administered after 6 weeks of polymicrobial infection. Periodontal disease parameters, including bacterial colonization/infection, immune response, inflammation, alveolar bone resorption, and systemic spread, were assessed post-euthanasia. All three periodontal pathogens colonized the rat oral cavity during polymicrobial infection. Polymicrobial infection induced an increase in total alveolar bone resorption, intrabony defects, and gingival inflammation. Treatment with bis-enoxacin significantly decreased alveolar bone resorption more effectively than either alendronate or doxycycline. Histologic examination revealed that treatment with bis-enoxacin and enoxacin reduced gingival inflammation and decreased apical migration of junctional epithelium. These data support the hypothesis that bis-enoxacin and enoxacin may be useful for the treatment of periodontal disease.

Figure 1. Serum Antibody Response: Serum IgG and IgM antibody levels were measured by ELISA.

Serum was collected post-euthanasia at 12 weeks following initial polymicrobial infection (n = 6). Each graph shows the results of serum IgG or IgM antibody levels when probing with either P. gingivalis, T.denticola or T. forsythia whole cell antigen. Asterisks indicate the following P-values when compared to controls *P<0.05; **P<0.005.

Figure 2. The area of bone resorption was measured in mm².

Analysis of the buccal and palatal/lingual surfaces of maxillae and mandibles were done post-euthanasia after 12 weeks of infection, including an overlapping 6 weeks of treatment. All groups were compared to Pg+Td+Tf (P. gingivalis + T. denticola + T. forsythia) group for statistical significance. Asterisks indicate the following P-values when compared to controls *P<0.05; **P<0.005; ***P<0.0001. Values are means ± standard deviations of the bone resorption measured. (i) Representative image of the area between the cemento-enamel junction (CEJ) and the alveolar bone crest (ABC) that is measured for bone resorption. (ii) Representative image with black arrows showing how intrabony defects were identified.

Figure 3. Comparative mandibular histology of alveolar bone sections from maxilla of rat infected with P. gingivalis/T. denticola/T. forsythia at 12 weeks.

Photomicrograph of the interdental area from rat jaws (all sections stained with hematoxylin and eosin and images captured at 200X magnification). A. Section from sham-infected rat displaying minimal inflammation and hyperplasia of the crevicular epithelium (E). B. C. and D. Representative sections from the infected-untreated rats exhibit mild to moderate inflammation and hyperplasia of the epithelium with elongation of the rete ridges (EH) and increase in small capillaries (BV). Apical junctional epithelium migration (EM) is noted in the Section C (black arrows). Dentin (D); Cementum (C); Connective tissue (CT); Cemento-enamel junction (CEJ); Alveolar bone crest (ABC).